Registration Dossier

Administrative data

Description of key information

Oral: NOAEL (rat): 1000 mg/kg body weight per day ; male/female, OECD TG 407, 2016

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-02-2014 to 19-01-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
other: US EPA OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents (July 2000)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) (31 March 2011)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: February 2014; signature: October 2015
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The species and strain was selected in accordance with the OECD TG 407 and the other relevant guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes.
- Age at study initiation: approximately 44 to 50 days.
- Weight at study initiation: males 198 - 242 g and females 152 - 207 g; individuals were randomly allocated to treatment groups.
- Fasting period before study: None
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Wood based bedding (certified) which was changed at appropriate intervals per week. Environmental enrichment was provided in the form of wooden chew blocks and plastic shelter. Cage distribution within the holding rack was randomized.
- Diet: Rodent Maintenance Diet (certified supplier), ad libitum (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).
- Water (e.g. ad libitum): ad libitum (except during urine collection)
- Acclimation period: 12 days.

DETAILS OF FOOD AND WATER QUALITY: Feed: Rodent Maintenance Diet – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2014-02-20 To: 2014-07-01
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. The test item was prepared at the appropriate concentrations in corn oil vehicle. The required amounts of test material were weighed out into a suitable container. Vehicle was added to the test item as applicable. The formulation was magnetically stirred until all test material was thoroughly mixed. The required volume was made up with vehicle and the formulation returned to the container and mixed using a magnetic stirrer until homogenous.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Corn oil was considered as appropriate based on test item solubility. The stability and homogeneity of the test item formulations were determined during the study. Results show the formulations to be homogeneous and stable for at least fifteen days when stored refrigerated and 1 day at ambient temperature. Stability was confirmed at concentrations of 1 and 200 mg/mL following storage at ambient temperature (nominally 21 ºC) for 24 hours and refrigerated (nominally 4 ºC) for up to 15 days. Formulations were therefore prepared weekly during the
treatment period, divided into daily aliquots and stored after one day at room temperature (nominally 21 ºC), refrigerated at nominally 4 ºC ºC in the dark.
- Concentration in vehicle: Samples of the test item formulations were taken and analyzed for concentration of test item (method of analysis provided in full study report). The results indicate that the prepared formulations were within ±6% of the nominal concentration. Corn oil formulations was assessed and confirmed at nominal concentrations, during refrigerated storage. The test item concentrations for each group are indicated in table 1.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group. For further information see 'Doses / concentrations'.
- Other: Dose-formulations were analysed during the study and were reported as with ± 10 to 15 % applied limits. The precision of the individual results from mean value was less than 2%. Confirming the precision of the analysis.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The homogeneity and stability was confirmed in corn oil formulations, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days.
- Refrigerated formulations were also analysed after refrigeration on receipt, Day 1, Day 8 and Day 15, a bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for 5 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.
- The analysis consisted of GC FID analysis with internal calibration (within a dedicated formulation analysis report attached to the full study report).
A representative sample of test formulation (1 mL, accurately weighed) and dissolved using swirling in a suitable volume of acetone. The extract was diluted using acetone, where necessary to provide a solution containing test item at an expected concentration of 40 μg/mL. Solutions also contained internal standard solution at 16 μg/mL, by adding the appropriate amount of internal standard to each sample before making to volume. The concentration of test item in the final solution was quantified by GC using FID detection as detailed in the chromatographic section. The analytical method was validated (details available within the full study report).
- Mean concentrations of dose-formulations analysed during the study were within ± 10 to 15% applied limits and % difference from mean were within 6% nominal confirming accurate test item/vehicle formulation.
Duration of treatment / exposure:
Minimum period 28 days followed by a 14 day recovery period (treatment free). The last dose was administered on Day 28.
Frequency of treatment:
Once daily at approximately the same time each day.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Recovery control group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low - Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate - Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High - Group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Recovery High - Group
No. of animals per sex per dose:
5 per sex per dose (5 male / 5 female)
Additional 5 animals per sex and group (control and high dose group only) were treated for 28 days and then allowed a 14-day treatment-free recovery period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on the results of a previously conducted 7-day sighting study (Report number attached to and cited in the full study report). Dose levels were selected following 7-day sighting test as: Group 1: 250 mg/kg/day, Group 2: 500 mg/kg/day Group 3: 1000 mg/kg/day (regulatory limit dose).I n the 7-day range finder (administered consecutively, for 7-days) determined: thee was no mortality, hair loss was observed in two males receiving 1000 mg/kg/day, this is considered likely to be a consequence of fighting and is not considered to be an effect of treatment. There was no effect of treatment on bodyweight gain, food consumption, water consumption, organ weights (kidney, liver or spleen), no macroscopic test item related lesions. The incidence and distribution of the dilated pelvis in the kidney of one female receiving 250 mg/kg/day and another at 1000 mg/kg/day was consistent with the common background seen in CD rats. Treatment with test item at dose levels of 250, 500 or 1000 mg/kg/day was well tolerated by both sexes and did not result in any evidence of toxicity. Basis: other: nominal in vehicle (Corn Oil)
- Rationale for animal assignment (if not random): Randomly assigned
- Post-exposure recovery period in satellite groups: 14 days.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All individuals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one to 2 hours after dosing. During the week 2 to termination and treatment free period, animals were observed daily. All observations were recorded. Additional functional observations were made as ‘additional evaluations’. Days 2, 3 and 4 to more closely monitor the condition of the animals and establish a pattern of signs. Signs were no longer observed at these additional time-points after Day 3 and, therefore, they were discontinued. In week 4, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to termination and, in the case of recovery group animals prior to termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study.

FOOD EFFICIENCY: No.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily. Water intake was observed daily, for each cage group, by visual inspection.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period (day 29) for all non-recovery test and control group individuals. End of recovery period (day 15; recovery phase) for all recovery group individuals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight.
- How many animals: All main study and recovery.
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices – including: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count – including: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT). Morphology: Anisocytosis, Macrocytosis, Microcytosis, , Hypochromasia, Hyperchromasia
Additionally: Prothrombin time (PT) was assessed and Activated partial thromboplastin time (APTT) was assessed using samples collected into sodium citrate solution

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period (day 29) for all non-recovery test and control group individuals. End of recovery period (day 15; recovery phase) for all recovery group individuals.
- Animals fasted: Yes, overnight.
- How many animals: All main study and recovery.
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Total bile acids (Bi Ac), Urea, Urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K),
Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (A/G Ratio) was calculated

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalytical investigations were performed on all non-recovery test and control group animals during day 29 and on all recovery group animals during days 14-15.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (food withheld during time of urine collection; overnight)
- Parameters checked: urine volume, urine appearance, urine density, pH, ketones, bilirubin, urobilnogen, blood pigments, protein, sodium, potassium, chloride, creatinine, glucose. Microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Casts, Spermatozoa, Other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Prior to the start of treatment and on Days 7, 14, 21 and 28, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- organs weighed: Adrenals, Liver, Brain, Ovaries, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Pituitary (post-fixation), Thyroid/Parathyroid (post fixation), Prostate and Seminal Vesicles, Uterus with Cervix (with coagulating glands and fluids)

HISTOPATHOLOGY: Yes
- Organs and tissues preserved in neutral buffered 10% formalin: Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and Rectum pons), Salivary glands (submaxillary), Caecum, Sciatic nerve, Colon, Seminal vesicles (with coagulating glands and fluids), Duodenum, Epididymides (Preserved in modified Davidson’s fluid), Skin, Esophagus, Spinal cord (cervical, mid thoracic and lumbar), Eyes (fixed in Davidson’s fluid), Gross lesions, Spleen, Heart, Stomach, Ileum ,Testes (Preserved in modified Davidson’s fluid), Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Trachea, Lungs (with bronchi) - inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative, Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus & Cervix, Mammary gland, Vagina, Muscle (skeletal).
Microscopic analysis was conducted thereof. Any macroscopically observed lesions were also processed.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Where appropriate, data transformations were performed using the most suitable method. Data were analysed using the decision tree from proprietary tables and statistics modules incorporating, homogeneity of variance from mean values was analysed using Bartlett’s test.
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. If Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead. Where there were only two groups, comparisons were made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied.
For grip strength, motor activity and clinical pathology data, if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom) were applied unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Clinical signs:
no effects observed
Description (incidence and severity):
There was no test item related clincal signs in relation to treatment.

Post dose salivation were evident during the first week of treatment; it was considered that these signs were from test item palatability of the dose administration rather than from toxicity.
Mortality:
no mortality observed
Description (incidence):
There was no test item related mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no test item related effect in bodyweight gain.

There were minor fluctuations outside the normal background ranges for main study and recovery animals of this age and strain.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
There was no test item related effect in food consumption.
Food efficiency:
no effects observed
Description (incidence and severity):
See body weight and weight changes sections.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There was no test item related water consumption changes.
Ophthalmological findings:
not examined
Description (incidence and severity):
There were no toxicologically significant reported effects to the eyes (in life or post termination) in the parameters examined.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day: haematological examination on Day 29 indicated, when compared with control values, statistically significant higher group mean monocyte and large unstained cell counts for females (1.9X control and 1.6X control, respectively) and a higher than control platelet count for males (1.3X control). All of the differences from control were no longer present following the recovery phase.

At 30 mg/kg bw/day: statistically significant lower than control mean cell volumes for males (0.96X control), however this was considered minor and lacked dose-relationship and was confined to one sex and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 and 300 mg/kg bw/day: on Day 29 revealed a lower than control group mean alanine amino-transferase activity was noted for both sexes (0.74X and 0.81X control respectively for males and 0.84X and 0.81X control respectively for females). The group mean alkaline phosphatase was lower than control for females receiving 300 or 1000 mg/kg/day (0.82X and 0.77X control respectively) but did not achieve statistical significance and aspartate amino transferase levels for females at 1000 mg/kg/day were lower than control (0.89X control) achieving statistical significance, and are considered to be treatment related. Higher than control group mean cholesterol and triglyceride were recorded for females treated with 1000 mg/kg/day (1.34X control and 2.0X control, respectively) with both values achieving statistical significance. Lower than control group mean chlorine values were recorded for males and females treated with 1000 mg/kg/day with both values achieving statistical significance and were considered to be treatment related.

At 1000 and 30 mg/kg bw/day: lower than control group mean bile acid concentration was recorded for females treated (0.58X control for both concentrations) and males receiving for males at 300 or 1000 mg/kg/day (0.38X control and 0.41X control respectively) appearing to be treatment related, however the review of the individual data revealed considerable variation in individual values and many of values were within control ranges and background control ranges.

All above were no longer present following the 14 day recovery period.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In all treatment groups: a lower group mean urine volume for both sexes (0.63X control, 0.33X control and 0.59X control respectively for males and 0.48X control, 0.37X control and 0.59X control respectively for females), a lower than control group mean pH value for all treated groups for both sexes (0.89X control, 0.81X control and 0.77X control respectively for males and 0.95X control, 0.88X control and 0.85X control respectively for females) with statistical significance achieved with the exception of the mean pH value for females at 30 mg/kg/day.

At 1000 and 300 mg/kg bw/day: higher than control group mean specific gravity was also seen for both sexes (1.03X control and 1.04X control respectively for males and 1.02X control and 1.02X control respectively for females) with statistical significance achieved in the males. Analysis also revealed a higher than control group mean protein level for all treated male groups (1.6X control, 2.0X control and 2.6X control respectively for males,) attaining statistical significance for 1000 mg/kg bw/day group. Lower than control group mean sodium level was noted for both sexes for all treated groups (0.74, 0.32X and 0.26X control for males respectively and 0.48X, 0.51X and 0.50X control for females respectively) with males attaining statistical significance.

All above were no longer present following the 14 day recovery period.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item related effect on sensory reactivity or grip strength.

Within motor activity at 1000 mg/kg bw/day: Week 4 motor activity investigations revealed higher than control group mean high beam (rearing activity) scores for males with statistical significance attained at the 24, 42 and 54 minute timepoints and for the total value for. High beam activity was also slightly higher than control for females, however the difference from control was small and attributed to biological variation. The values were considered to be within the laboratory background range and therefore unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In all treatment groups: after 4 weeks of treatment, group mean body weight adjusted kidney weights were statistically significant higher than control for all treated groups in males (1.11X control, 1.12X control and 1.29X control respectively for males). This difference was no longer observed following the recovery period.

At 1000 and 300 mg/kg bw/day: group mean body weight adjusted liver weights were statistically significantly higher than control for females and males receiving 1000 mg/kg/day (1.27X control for males and 1.11X control and 1.18X control respectively for females). After 2 weeks of recovery, group mean body weight adjusted liver weights remained statistically significantly higher than control for females (1.19X control), however, there was evidence of recovery in the males.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic lesions in treatment or recovery groups.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day: kidneys: treatment related changes were seen in the kidneys of males and females and included hyaline droplets.

After two weeks recovery: not examined.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related abnormalities detected.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested
Remarks:
The kidney and liver findings were considered either adaptive or have no relevance to human health.
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for males and females is defined as 1000 mg/kg body weight per day in males and females; since the kidney and liver findings were considered either adaptive or have no relevance to human health.
Executive summary:

The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Following a previously conducted 7-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 30, 300 or 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Corn Oil) at a dose volume of 5 mL/kg. Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations including sensory reactivity, grip strength and motor activity were performed, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination and at termination. Histopathological examination of selected tissues was performed. There was no test item related effect on mortalities, clinical signs, effect on sensory reactivity, grip strength or motor activity, bodyweight gain, food consumption and water consumption. Haematological examination revealed high group mean monocyte and large unstained cell counts for females receiving 1000 mg/kg/day and a higher than control platelet count for males receiving 1000 mg/kg/day. All haematological parameters were similar to control at the end of the recovery period. In clinical chemistry: low bile acid concentration for females treated with 30 or 1000 mg/kg/day and males receiving 300 or 1000 mg/kg/day and high cholesterol and triglyceride values for females at 1000 mg/kg/day. However alanine amino-transferase, alkaline phosphatase and aspartate amino transferase levels were slightly lower than controls for these groups. All blood chemistry parameters had returned to control levels at the end of the recovery period. In urinalysis: low urine volumes, pH and sodium values for all treated male and female groups. A higher than control specific gravity was also seen for both sexes receiving 300 or 1000 mg/kg/day and a high protein level was recorded for all treated male groups. All urine parameters were similar to control at the end of the recovery period. In organ weights: higher than control liver weights were recorded for females receiving 300 or 1000 mg/kg/day and males receiving 1000 mg/kg/day, higher than control kidney weights were recorded for all treated groups in males, with the exception of liver weights for females, these parameters had returned to control levels at the end of the recovery period. Macroscopic changes related to treatment with test item were seen in the kidney: hyaline droplets were evident in males treated at 1000 mg/kg/day after 28 days of administration. The presence of hyaline droplets, also known as intra-cytoplasmic protein droplets, in the cortical tubules of male kidneys is the early indicator of hydrocarbon neuropathy. The droplets are considered to be the result of reversible binding of test item to α2-microglobulin leading to accumulation within the tubular cell lysoma in the kidney. α2-microglobulin is not present in humans. This finding is not considered relevant to human health. The oral (gavage) administration of the test item to males/females at dose levels of 30, 300 or 1000 mg/kg bw/day resulted in adaptive liver changes (disturbances in liver enzymes, serum cholesterol and triglyceride concentration and increased liver weights) with evidence of recovery. Kidney findings were considered to have no relevance to human health. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day for males/females.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of a high quality (Klimisch 1); The available information as a whole meets the tonnage driven information requirements of REACH.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose - Oral:

Key study : OECD TG 407, 2016 : The study was performed according the requirements of OECD TG 407, EU method B.7, US EPA OPTTS 870.3050 and Japan MHLW, METI and MOE guidelines under GLP conditions. Following a previously conducted 7-day sighting study, the systemic toxic potential of the test item was assessed orally in a 28 day gavage study in Crl:CD(SD) rats. Recovery from any effects was evaluated during a subsequent 14 day recovery period. Three groups, each comprising five male and five female CD rats, received test item at doses of 30, 300 or 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Corn Oil) at a dose volume of 5 mL/kg. Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days. Clinical signs, functional observations including sensory reactivity, grip strength and motor activity were performed, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment free period. All individuals were subjected to gross necropsy examination and at termination. Histopathological examination of selected tissues was performed. There was no test item related effect on mortalities, clinical signs, effect on sensory reactivity, grip strength or motor activity, bodyweight gain, food consumption and water consumption. Haematological examination revealed high group mean monocyte and large unstained cell counts for females receiving 1000 mg/kg/day and a higher than control platelet count for males receiving 1000 mg/kg/day. All haematological parameters were similar to control at the end of the recovery period. In clinical chemistry: low bile acid concentration for females treated with 30 or 1000 mg/kg/day and males receiving 300 or 1000 mg/kg/day and high cholesterol and triglyceride values for females at 1000 mg/kg/day. However alanine amino-transferase, alkaline phosphatase and aspartate amino transferase levels were slightly lower than controls for these groups. All blood chemistry parameters had returned to control levels at the end of the recovery period. In urinalysis: low urine volumes, pH and sodium values for all treated male and female groups. A higher than control specific gravity was also seen for both sexes receiving 300 or 1000 mg/kg/day and a high protein level was recorded for all treated male groups. All urine parameters were similar to control at the end of the recovery period. In organ weights: higher than control liver weights were recorded for females receiving 300 or 1000 mg/kg/day and males receiving 1000 mg/kg/day, higher than control kidney weights were recorded for all treated groups in males, with the exception of liver weights for females, these parameters had returned to control levels at the end of the recovery period. Macroscopic changes related to treatment with test item were seen in the kidney: hyaline droplets were evident in males treated at 1000 mg/kg/day after 28 days of administration. The presence of hyaline droplets, also known as intra-cytoplasmic protein droplets, in the cortical tubules of male kidneys is the early indicator of hydrocarbon neuropathy. The droplets are considered to be the result of reversible binding of test item to α2-microglobulin leading to accumulation within the tubular cell lysoma in the kidney. α2-microglobulin is not present in humans. This finding is not considered relevant to human health. The oral (gavage) administration of the test item to males/females at dose levels of 30, 300 or 1000 mg/kg bw/day resulted in adaptive liver changes (disturbances in liver enzymes, serum cholesterol and triglyceride concentration and increased liver weights) with evidence of recovery. Kidney findings were considered to have no relevance to human health. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day for males/females.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for specific organ toxicity repeated exposure (STOT-RE).

Since there was no reported significant effects relevant to humans reported at guidance related levels (ORAL ≤ 300 mg/kg bw/day) then there is no requirement to classify STOT-RE.

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017), Section 3.9.2 : Table 3.16 - Equivalent guidance values for 28-day and 90-day studies